CN101003822A - Method for producing D amino acid by immobilizing acylation enzyme of penicillin - Google Patents
Method for producing D amino acid by immobilizing acylation enzyme of penicillin Download PDFInfo
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Abstract
This invention discloses a method for preparing D-amino acid. The method comprises: derivatizing DL-amino acids to obtain N-phenylacetyl-DL-amino acids, performing enzyme-catalyzed asymmetric hydrolysis in aqueous solution to obtain N-phenylacetyl-D-amino acid, and performing chemical hydrolysis and crystallization to obtain D-amino acid. The enzyme used is immobilized penicillin acylase, which can be used for more than 100 times. The method has such advantages as high yield and high product optical purity, and is suitable for the majority of DL-amino acids resolution.
Description
Technical field
The present invention relates to the amino acid whose production technique of a kind of D-.Specifically, the present invention relates to utilize the immobilized penicillin acylated enzyme enzyme digestion reaction, generate the amino acid whose production technique of D-.
Background technology
Amino acid is to contain amino organic acid, is to constitute proteinic fundamental unit.20 kinds of primary amino acids forming living matter in the human body all have two kinds of enantiomorph D-amino acid and L-amino acid that are mutually mirror image except that glycine.Have constantly held and only have L-amino acid in the living matter, development along with analytical procedure, people have found various D-amino acid in succession in marine animal, terrestrial animal, vertebra and invertebrates, spermatophyte and human body, D-amino acid has just caused people's attention, and recognize that D-amino acid has the irreplaceable status of L-amino acid in vital movement and medication preparation, particularly as the intermediate of β-Nei acyl acyl class microbiotic and physiologically active peptide.
Amino acid whose function of D-and physiological action are just illustrated gradually, and some human disease such as cataract, early the amino acid whose content of D-is closely related in dementia, nephropathy etc. and the body, so D-amino acid is very important to study of disease and old and feeble Study on Mechanism.Experimentation on animals proves that the D-amino acid of excess can be blocked the synthetic of some important biomolecule materials, thereby suppresses the growth of cell, organism.The polypeptide that D-amino acid constitutes usually can not or can only be lentamente by the peptide enzymic hydrolysis, so D-amino acid has been used to synthetic β-Nei acyl acyl class microbiotic and physiologically active peptide more and more.
D-amino acid is non-protein source amino acid, can not adopt fermentative Production as L-amino acid.About the amino acid whose preparation of D-, the method for bibliographical information mainly contains induced crystallization method, chemical resolution method, asymmetric transformation approach, biological process etc.
According to Susenm Tatsumi etc., in saturated or super-saturated racemize Xie Ansuan (DL-Val) hydrochloride solution, add crystal seed, can go out D-Xie Ansuan hydrochloride by preferential crystallization, in and D-Xie Ansuan hydrochloride obtain the D-Xie Ansuan, optical purity is 93%, and yield is lower than 10%.The induced crystallization legal system get the D-amino acid cycle long, yield is low, optical purity is not high yet, is not suitable for industrial production.
Chemical resolution method mainly is to utilize the resolution reagent of chirality and DL-amino acid to form diastereoisomeric salt, by fractional crystallization it is separated, and diastereoisomeric salt obtains optically active D-amino acid by taking off resolution reagent with acid or alkali neutralization reaction.Product yield and optical purity that chemical resolution method obtains are not high enough, and chiral selectors costs an arm and a leg, and have limited large-scale industrial production.
Asymmetric conversion method directly prepares D-amino acid by asymmetric conversion by racemic modification or L-amino acid as a kind of new method for splitting of optically active compound.But expensive chiral source, chiral auxiliary(reagent) or chiral catalyst are adopted in chemical asymmetric synthesis, and resolution process route complexity, production cost height have only the D-amino acid of minority to produce with this method.
The method that biological process production D-amino acid relates to has enzyme catalysis Split Method and enzyme catalysis conversion method.The enzyme catalysis Split Method mainly comprises acidylate enzyme process and carboxypeptidase method.Japan utilized immobilization L-L-Aminoacylase in 1969, was succeedd by N-acetyl-DL-amino acid continuous production L-amino acid.This technology also has been used for the amino acid whose commercial mass production of D-; basic technology is to split the N-acetyl-DL-amino acid with immobilized L-L-Aminoacylase; L-amino acid is separated out by precipitation; acetylize then; racemization becomes the N-acetyl-DL-amino acid; recirculation splits, and the N-acetyl-D-amino acid of generation prepares D-amino acid through hydrolysis.The main drawback that exists is that the end product productive rate is low, process is complicated, production cost is high.More domestic manufacturers adopt pig or ox kidney L-L-Aminoacylase to prepare D-amino acid, but the enzyme source is limited, the production cost height.The L-carboxypeptidase method of Holland DSM N. V. exploitation prepares the large-scale production that D-amino acid technology successfully is used for semisynthetic antibiotics side chain D-phenylglycine and D-glycin, and this technology universality is poor, is not suitable for the amino acid whose production of other D-.The enzyme catalysis conversion method mainly comprises microbial degradation method, D-amino acid transaminase method and using hydantoinase etc.Microbial degradation method employing microbial enzyme or whole cell are the L-enantiomorph in the biological catalyst degradation DL-amino acid, and D-amino acid keeps.The shortcoming of this method is that the amino acid whose theoretical yield of D-only is 50%, and optical purity is difficult to reach requirement.D-amino acid transaminase method is a raw material with the ketone acid that is difficult to prepare, and expensive D-amino acid is the ammonia donor, and preparation cost is very high.The using hydantoinase that Japan takes the lead in developing is a raw material with the glycolylurea of chemosynthesis, prepares D-amino acid by a bacterium double-enzyme method, two bacterium double-enzyme method or an enzyme one acid system, and this method successfully is used for the kiloton large-scale production of D-phenylglycine and D-glycin.Domestic National Engineering Research Center for Biotechnology adopts this legal system to be equipped with D-phenylalanine, D-tyrosine, D-L-Ala, D-Xie Ansuan etc., but the preparation cost height of glycolylurea has limited the large-scale application of this method in D-amino acid is produced.
Summary of the invention
The objective of the invention is at the deficiency on the prior art, propose a kind of immobilized enzyme that utilizes and produced the amino acid whose technology of D-, use this method to split DL-amino acid products obtained therefrom yield height, the optical purity height is suitable for the fractionation of most DL-amino acid enantiomers.
The present invention is achieved by following proposal: the amino acid whose method for splitting of a kind of DL-comprises the steps:
(1) the amino acid whose preparation of N-phenylacetyl-DL-: the synthetic N-phenylacetyl of DL-amino acid and phenyllacetyl chloride reaction under agitation-DL-amino acid,
The reaction mol ratio of DL-amino acid and phenyllacetyl chloride is 1: (1-1.5), temperature of reaction is 0-40 ℃, and reaction medium is a NaOH solution, and the reaction times is 1-12 hour, add concentrated hydrochloric acid adjust pH 0-3, suction filtration gets white solid I, and filtrate concentrates, cooling is filtered, and obtains white solid II.
(2) enzymolysis N-phenylacetyl-DL-amino acid: under agitation white solid I and II are put in the borate buffer of 0.05mol/L,
Concentration of substrate 0.1-1mol/L adds 100 immobilized penicillin acylated enzymes that restrain, and stirring reaction 2-12 hour, temperature of reaction 20-40 ℃, reaction pH=6-10, stir speed (S.S.) is 100-300 rev/min.Centrifugation goes out immobilized enzyme, transfers pH=0-4 with concentrated hydrochloric acid, and filtration gets white solid III, and filtrate concentrates, and cools off, and filtration gets white solid IV, washes solid III and IV to not containing L-amino acid, gets white solid V, and merging filtrate and washing lotion get solution IV.Immobilized penicillin acylated enzyme is used for repeating next time splitting.
(3) hydrolyzing N-phenylacetyl-D-amino acid: white solid V is joined in the 6mol/L HCl solution, stirring reaction under heating condition,
Temperature of reaction is 100-140 ℃, and the reaction times is 1-6 hour, concentrates, and is cooled to 4 ℃, and suction filtration gets white crystal VII and filtrate VII.
(4) with filtrate VII NH
3Water is transferred pH=3-10, spends the night 4 ℃ of refrigerations, and crystallization is filtered, and washing gets white crystal IX, filtrate
Concentrate, 4 ℃ down refrigeration spend the night, crystallization is filtered, white crystal X and filtrate XI, merge crystal IX and crystal X, be dissolved in the mixed solvent of second alcohol and water, ethanol/water=10/1-5/1, recrystallization,, filter also washing, obtain D-amino acid.Filtrate XI is directly used in next recrystallization.
In terms of existing technologies, this method for splitting has and splits route weak point, process stabilizing, simple to operate, good separating effect, product purity height, and production cost is low.Only ethanol, whole process greenization with an organic solvent in the inventive method production process in addition.
The enzyme that the present invention uses is easy to get as the immobilized penicillin acylated enzyme of domestic production, enzyme source, fully can large-scale industrial production, and this immobilized enzyme repeats to split number of times and can reach more than 100 times in addition, greatly reduces production cost.
The chemical equation that the D-amino acid that the present invention relates to is produced is as follows:
Description of drawings
Fig. 1 represents D-amino acid technological process of production figure.
Embodiment
Embodiment one: the production technique of D-methionine(Met)
1. synthetic N-phenylacetyl-DL-methionine:
In 10 liters reactor, add 298 gram DL-methionine, 240 gram NaOH, 4000 ml waters, fully stir down at 10 ℃, stir and drip 387.5 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 5 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (240 milliliters) into original volume, and the white solid of separating out is filtered in cooling, merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-methionine
The white solid of previous step joined in 10 liters the reactor; add 4000 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 100 grams; at 30 ℃ of following stirring reaction 5h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1-2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (200 milliliters) into original volume, filters; solid washes with water to not containing L-methionine(Met) (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-methionine(Met)
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6mol/LHCl solution, at 110 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, tabular crystal toluylic acid directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-methionine(Met):
Filtrate is used NH
3Water is transferred pH=5.74, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-methionine(Met) 120 gram (productive rate 40%, α
D 20=-22.5 (C=2,1NHCl).Filtrate is directly used in next recrystallization.
Embodiment two: the production technique of D one L-Ala
1. synthetic N-phenylacetyl-DL-L-Ala:
In 5 liters reactor, add 365 gram DL-L-Ala, 340 gram NaOH, 2000 ml waters fully stir down at 10 ℃, stir down
Drip 697.5 gram phenyllacetyl chlorides, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 5 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (100 milliliters) into original volume, and the white solid of separating out is filtered in cooling, merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-L-Ala
The white solid of previous step joined in 10 liters the reactor; add 6800 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 140 grams; at 30 ℃ of following stirring reaction 5h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1-2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (340 milliliters) into original volume, filters; solid washes with water to not containing L-L-Ala (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-L-Ala
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6mol/L HCl solution, at 110 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, tabular crystal toluylic acid directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-L-Ala:
Filtrate is used NH
3Water is transferred pH=6.11, spends the night 4 ℃ of refrigerations, and the crystal that filter is separated out, filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-L-Ala 140 gram (productive rate 40%, α
D 20=-14.6 ° (C=2,5N HCl).Filtrate is directly used in next recrystallization.
Embodiment three: the production technique of D-L-glutamic acid
1. synthetic N-phenylacetyl-DL-L-glutamic acid:
In 5 liters reactor, add 500 gram DL-L-glutamic acid, 460 gram NaOH, 3000 ml waters, fully stir down at 10 ℃, stir Dropwise 5 73.5 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 5 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (150 milliliters) into original volume, and the white solid of separating out is filtered in cooling, merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-L-glutamic acid
The white solid of previous step joined in 10 liters the reactor; add 4200 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 200 grams; at 30 ℃ of following stirring reaction 5h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1-2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (210 milliliters) into original volume, filters; solid washes with water to not containing L-L-glutamic acid (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-L-glutamic acid
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6mol/L HCl solution, at 110 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, tabular crystal toluylic acid directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-L-glutamic acid:
Filtrate is used NH
3Water is transferred pH=3.15, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-L-glutamic acid 140 gram (productive rate 40%, α
D 20=-31.4 ° (C=2,2N HCl).Filtrate is directly used in next recrystallization.
Embodiment four: the production technique of D-aspartic acid
1. synthetic N-phenylacetyl-DL-aspartic acid:
In 5 liters reactor, add 400 gram DL-aspartic acids, 600 gram NaOH, 1500 ml waters, fully stir down at 10 ℃, stir and drip 620 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 5 hours, stopped reaction, add concentrated hydrochloric acid and transfer pH=1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m,, 60 ℃ of temperature) and makes it 1/10 (150 milliliters) into original volume, and the white solid of separating out is filtered in cooling, merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-aspartic acid
The white solid of previous step joined in 10 liters the reactor; add 4200 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 200 grams; at 30 ℃ of following stirring reaction 4.5h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1-2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (210 milliliters) into original volume, filters; solid washes with water to not containing L-aspartic acid (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-aspartic acid
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 600 milliliters of 6mol/L HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (120 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, tabular crystal toluylic acid directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 120 milliliters.
4. crystallization and recrystallization D-aspartic acid:
Filtrate is used NH
3Water is transferred pH=2.85, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-aspartic acid 160 gram (productive rate 40%, α
D 20=-24.7 ° (C=2,6NHCl).Filtrate is directly used in next recrystallization.
Embodiment five: the production technique of D-phenylalanine
1. synthetic N-phenylacetyl-DL-phenylalanine:
In 5 liters reactor, add 330 gram DL-phenylalanines, 180 gram NaOH, 2500 ml waters, fully stir down at 10 ℃, stir and drip 287.5 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (125 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-phenylalanine
The white solid of previous step joined in 10 liters the reactor; add 7000 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 130 grams; at 30 ℃ of following stirring reaction 4h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1～2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (350 milliliters) into original volume, filters; solid washes with water to not containing L-phenylalanine (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-phenylalanine
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6mol/L HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, tabular crystal toluylic acid directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-phenylalanine:
Filtrate is used NH
3Water is transferred pH=5.49, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-phenylalanine 150 gram (productive rate 40%, α
D 20=34.5 ° of (C=2, H
2O).Filtrate is directly used in next recrystallization.
Embodiment six: the production technique of D-phenylglycine
1. synthetic N-phenylacetyl-DL-phenylglycine:
In 5 liters reactor, add 302 gram DL-phenylglycines, 180 gram NaOH, 2500 ml waters, fully stir down at 10 ℃, stir and drip 287.5 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (125 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-phenylglycine
The white solid in last-step is joined in 10 liters the reactor; add 7000 milliliters of 0.05 mole of borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 130 grams; at 30 ℃ of following stirring reaction 4h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1～2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (350 milliliters) into original volume, filters; solid washes with water to not containing L-phenylalanine (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-phenylglycine
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 4.5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-phenylglycine:
Filtrate is used NH
3Water is transferred pH=5.6, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-phenylglycine 127 gram (productive rate 42%, α
D 20=-31.5 ° (C=, 1NHCl).Filtrate is directly used in next recrystallization.
Embodiment seven: the leucic production technique of D-
1. synthetic N-phenylacetyl-DL-leucine:
In 5 liters reactor, add 392 gram DL-leucines, 320 gram NaOH, 3000 ml waters, fully stir down at 10 ℃, stir and drip 620 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (150 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-leucine
The white solid of previous step joined in 10 liters the reactor; add 7500 milliliters of 0.05 mole of borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 200 grams; at 30 ℃ of following stirring reaction 4h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1～2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (375 milliliters) into original volume, filters; solid washes with water to not containing L-leucine (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-leucine
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 800 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (160 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 160 milliliters.
4. crystallization and recrystallization D-leucine:
Filtrate is used NH
3Water is transferred pH=6.01, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-leucine 156 gram (productive rate 40%, α
D 20=-17 ° (C=2,6NHCl).Filtrate is directly used in next recrystallization.
5. repeat to split the L-leucine that second step produced
Embodiment eight: the production technique of D-Xie Ansuan
1. synthetic N-phenylacetyl-DL-valine:
In 5 liters reactor, add 468 gram DL-valine, 360 gram NaOH, 3000 ml waters, fully stir down at 10 ℃, stir and drip 697.5 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (150 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-valine
The white solid of previous step joined in 20 liters the reactor; add 10000 milliliters of 0.05 mole of borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 200 grams; at 30 ℃ of following stirring reaction 4h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1～2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (500 milliliters) into original volume, filters; solid washes with water to not containing L-Xie Ansuan (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-Xie Ansuan
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 700 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (120 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 120 milliliters.
4. crystallization and recrystallization D-Xie Ansuan:
Filtrate is used NH
3Water is transferred pH=6, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-Xie Ansuan 187 gram (productive rate 40%, α
D 20=-28.9 ° (C=2,5NHCl).Filtrate is directly used in next recrystallization.
Embodiment nine: the production technique of D-Methionin
1. synthetic N, N
ε-two phenylacetyls-DL-Methionin:
In 5 liters reactor, add 146 gram DL-Methionins, 160 gram NaOH, 2000 ml waters, fully stir down at 10 ℃, stir and drip 620 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/10 (200 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N, N
ε-two phenylacetyls-DL-Methionin
The white solid of previous step joined in 10 liters the reactor, add 5000 milliliters of 0.05 mole of borate buffers, transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 200 grams; at 30 ℃ of following stirring reaction 12h, stopped reaction, centrifugation goes out enzyme; solution is transferred pH=5.5 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (250 milliliters) into original volume, filters; solid washes with water to not containing L-Methionin (HPLC method), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N, N
ε-two phenylacetyls-DL-Methionin
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-Methionin:
Filtrate is used NH
3Water is transferred pH=9.6, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-Methionin 51 gram (productive rate 35%, α
D 20=-21 ° (C=1,6NHCl).Filtrate is directly used in next recrystallization.
Embodiment ten: the production technique of D-ornithine
1. synthetic N, N
ε-two phenylacetyls-DL-ornithine:
In 5 liters reactor, add 203 gram DL-ornithine dihydrochlorides, 160 gram NaOH, 1000 ml waters, fully stir down at 10 ℃, stir and drip 372 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 12 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/10 (100 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N, N
ε-two phenylacetyls-DL-ornithine
The white solid of previous step joined in 10 liters the reactor, add 5000 milliliters of 0.05 mole of borate buffers, transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 200 grams; at 30 ℃ of following stirring reaction 12h, stopped reaction, centrifugation goes out enzyme; solution is transferred pH=5.5 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (250 milliliters) into original volume, filters; solid washes with water to not containing L-Methionin (HPLC method), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N, N
ε-two phenylacetyls-DL-Methionin
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 400 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (80 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 80 milliliters.
4. crystallization and recrystallization D-ornithine:
Filtrate is used NH
3Water is transferred pH=9.6, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-ornithine 46.2 gram (productive rate 35%, α
D 20=-28.4 ° (C=2,5NHCl).Filtrate is directly used in next recrystallization.
Embodiment 11: the production technique of D-Terleu
1. synthetic N-phenylacetyl-DL-Terleu:
In 5 liters reactor, add 262 gram DL-Terleus, 200 gram NaOH, 1000 ml waters, fully stir down at 10 ℃, stir and drip 400 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/10 (100 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-Terleu
The white solid of previous step joined in 10 liters the reactor; add 5000 milliliters of 0.05 mole of borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 220 grams; at 30 ℃ of following stirring reaction 6h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1～2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (250 milliliters) into original volume, filters; solid washes with water to not containing L-Terleu (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-Terleu
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 500 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (100 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 160 milliliters.
4. crystallization and recrystallization D-Terleu:
Filtrate is used NH
3Water is transferred pH=6.1, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-Terleu 156 gram (yield 40%, α
D 20=-6.92 ° (C=2,5NHCl).Filtrate is directly used in next recrystallization.
Embodiment 12: D-is to the production technique of fluorophenyl glycine
1. synthetic N-phenylacetyl-DL-is to fluorophenyl glycine:
In 5 liters reactor, add 169 gram DL-to fluorophenyl glycine, 110 gram NaOH, 1300 ml waters, fully stir down at 10 ℃, stir and drip 180 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/10 (130 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-is to fluorophenyl glycine
The white solid of previous step joined in 10 liters the reactor; add 5000 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 120 grams; at 30 ℃ of following stirring reaction 6h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1～2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/20 (250 milliliters) into original volume, filters; solid washes with water to not containing L-to fluorophenyl glycine (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-is to fluorophenyl glycine
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 350 milliliters of 6 moles of HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (100 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, crystal toluylic acid on chip directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 100 milliliters.
4. crystallization and recrystallization D-are to fluorophenyl glycine:
Filtrate is used NH
3Water is transferred pH=6.4, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-to fluorophenyl glycine 68 gram (yield 40%, α
D 20=-137.5 ° (C=2,1NHCl).Filtrate is directly used in next recrystallization.
Embodiment 13: the production technique of D-o-chlorobenzene glycine
1. synthetic N-phenylacetyl-DL-o-chlorobenzene glycine:
In 10 liters reactor, add 370 gram DL-o-chlorobenzene glycines, 240 gram NaOH, 4000 ml waters, fully stir down at 10 ℃, stir and drip 372 gram phenyllacetyl chlorides down, control pH=8-10 in the reaction process, in 10 ℃ of the temperature, be added dropwise to complete back insulation reaction 2 hours, at room temperature continued stirring reaction then 6 hours, stopped reaction, add concentrated hydrochloric acid pH is transferred to 1-2, separate out a large amount of white solids, vacuum filtration obtains white solid.Filtrate decompression concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) and makes it 1/20 (200 milliliters) into original volume, cooling, and the white solid that vacuum filtration is separated out merges the white solid that obtains for twice, the input the next step.
2. enzymolysis N-phenylacetyl-DL-o-chlorobenzene glycine
The white solid of previous step joined in 20 liters the reactor; add 10000 milliliters of 0.05mol/L borate buffers; transfer pH=8.0 with NaOH; the immobilized penicillin acylated enzyme that adds 150 grams; at 30 ℃ of following stirring reaction 6h; stopped reaction; centrifugation goes out enzyme; solution is transferred pH=1-2 with concentrated hydrochloric acid; vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) makes it 1/30 (330 milliliters) into original volume, filters; solid washes with water to not containing L-o-chlorobenzene glycine (ninhydrin), directly drops into for the 3rd step.Filtrate, washing lotion were dropped into for the 5.1st step.
Annotate: immobilized penicillin acylated enzyme can reuse repeatedly, when enzyme live for beginning 1/2 the time stop using.
3. hydrolyzing N-phenylacetyl-D-o-chlorobenzene glycine
The 2nd white solid that obtains of step joined in 1 liter the reactor, add 600 milliliters of 6mol/L HCl solution, at 120 ℃ of following stirring reaction 5h, vacuum concentration (vacuum tightness 0.1p.m, 60 ℃ of temperature) make it 1/5 (120 milliliters) into original volume, be cooled to 4 ℃, vacuum filtration, tabular crystal toluylic acid directly dropped into for the 5.2nd step.Filtrate put into for the 4th step for 120 milliliters.
4. crystallization and recrystallization D-o-chlorobenzene glycine:
Filtrate is used NH
3Water is transferred pH=6.5, spends the night 4 ℃ of refrigerations, filters the crystal of separating out, and filtrate concentrates (vacuum tightness 0.1p.m, 60 ℃ of temperature) to 20 milliliters, 4 ℃ of following refrigerations are spent the night, and vacuum filtration merges the gained crystal twice, 95% ethyl alcohol recrystallization obtains D-o-chlorobenzene glycine 148 gram (yield 40%, α
D 20=-112.4 ° (C=2,1NHCl).Filtrate is directly used in next recrystallization.
Claims (10)
1. amino acid whose preparation method of D-, it is characterized in that: with DL-amino acid is raw material, derives to obtain N-phenylacetyl-DL-amino acid, uses the enzyme catalysis asymmetric hydrolysis under the aqueous solution, obtains N-phenylacetyl-D-amino acid, the Separation and Recovery enzyme; By chemical hydrolysis, crystallization obtains D-amino acid with gained N-phenylacetyl-D-amino acid.
2. the amino acid whose preparation method of D-according to claim 1 is characterized in that: described DL-amino acid is that DL-methionine, DL-L-Ala, DL-L-glutamic acid, DL-aspartic acid, DL-phenylalanine, DL-phenylglycine, DL-leucine, DL-valine, DL-Methionin, DL-ornithine, DL-Terleu, DL-are to fluorophenyl glycine, DL-o-chlorobenzene glycine.
3. the amino acid whose preparation method of D-according to claim 1 is characterized in that: described D-amino acid is that D-methionine(Met), D-L-Ala, D-L-glutamic acid, D-aspartic acid, D-phenylalanine, D-phenylglycine, D-leucine, D-Xie Ansuan, D-Methionin, D-ornithine, D-Terleu, D-are to fluorophenyl glycine, D-o-chlorobenzene glycine.
4. the amino acid whose preparation method of D-according to claim 1, it is characterized in that: under 0-40 ℃, NaOH solution is reaction medium, the synthetic N-phenylacetyl of DL-amino acid and phenyllacetyl chloride reaction under agitation-DL-amino acid, and the mol ratio of DL-amino acid and phenyllacetyl chloride is 1: (1-1.5).
5. the amino acid whose preparation method of D-according to claim 1 is characterized in that: enzyme is an immobilized penicillin acylated enzyme.
6. the amino acid whose preparation method of D-according to claim 1, it is characterized in that: in the enzyme catalysis asymmetric hydrolysis, concentration of substrate 0.1-1mol/L, reaction times 2-12 hour, temperature of reaction 20-40 ℃, reaction pH=6-10, stir speed (S.S.) is 100-300 rev/min, enzyme is 0.1-10 with N-phenylacetyl-DL-amino acid masses ratio: 1.
7. the amino acid whose preparation method of D-according to claim 1 is characterized in that: N-phenylacetyl-D-chemistry of amino acids is hydrolyzed to the mineral acid acid hydrolysis, and inorganic acid concentration is 3-6mol/L, and mineral acid is hydrochloric acid, sulfuric acid, phosphoric acid.
8. the amino acid whose preparation method of D-according to claim 1 is characterized in that: the temperature of N-phenylacetyl-D-chemistry of amino acids hydrolysis is 100-140 ℃, and the reaction times is 1-6 hour.
9. the amino acid whose preparation method of D-according to claim 1, it is characterized in that: N-phenylacetyl-D-amino acid is transferred to iso-electric point through the amino acid whose solution of D-that chemical hydrolysis obtains with alkali, refrigerate 8-12 hour, crystallization, used alkali are ammoniacal liquor, ammonia, triethylamine, sodium hydroxide, potassium hydroxide.
10. the amino acid whose preparation method of D-according to claim 1, it is characterized in that: the D-amino acid that obtains is passed through recrystallization purifying, the used solvent of purifying is the mixture of water and organic solvent, organic solvent is a kind of or any mixture in methyl alcohol, ethanol, propyl alcohol, Virahol, propyl carbinol and the acetone, and water that purifying is used and volume of organic solvent ratio are: (10-5): 1.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101864464A (en) * | 2010-04-29 | 2010-10-20 | 重庆凯乐尔生物催化技术有限公司 | Chemical-enzyme method for preparing (S)-2-chlorophenyl glycine methyl ester clopidogrel chiral intermediate |
| CN102352389A (en) * | 2011-09-29 | 2012-02-15 | 重庆邮电大学 | Chemical enzyme method for preparing L-para fluobenzene glycine |
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| CN101864464A (en) * | 2010-04-29 | 2010-10-20 | 重庆凯乐尔生物催化技术有限公司 | Chemical-enzyme method for preparing (S)-2-chlorophenyl glycine methyl ester clopidogrel chiral intermediate |
| CN102533888A (en) * | 2010-12-29 | 2012-07-04 | 浙江九洲药物科技有限公司 | Continuous enzymatic method for producing L-tert-leucine |
| CN102533888B (en) * | 2010-12-29 | 2015-07-15 | 浙江九洲药物科技有限公司 | Continuous enzymatic method for producing L-tert-leucine |
| CN102352389A (en) * | 2011-09-29 | 2012-02-15 | 重庆邮电大学 | Chemical enzyme method for preparing L-para fluobenzene glycine |
| CN102352388A (en) * | 2011-09-29 | 2012-02-15 | 重庆邮电大学 | Chemical enzyme method for preparing L-2-amino adipic acid |
| CN102352392A (en) * | 2011-09-29 | 2012-02-15 | 重庆邮电大学 | Chemical-enzyme method for preparing D-tyrosine |
| CN102392061A (en) * | 2011-09-29 | 2012-03-28 | 重庆邮电大学 | Chemical-enzyme method for preparing D-basic amino acid hydrochloride |
| CN102432413A (en) * | 2011-09-29 | 2012-05-02 | 重庆邮电大学 | Purification method for improving chiral purity of D-amino acid |
| CN102432413B (en) * | 2011-09-29 | 2014-02-19 | 重庆邮电大学 | Purification method for improving chiral purity of D-amino acid |
| CN102352388B (en) * | 2011-09-29 | 2014-10-15 | 重庆邮电大学 | Chemical enzyme method for preparing L-2-amino adipic acid |
| CN106083675A (en) * | 2016-06-03 | 2016-11-09 | 宁夏紫光天化蛋氨酸有限责任公司 | A kind of methionine novel crystal forms I and preparation method thereof |
| CN106083675B (en) * | 2016-06-03 | 2018-07-27 | 宁夏紫光天化蛋氨酸有限责任公司 | A kind of methionine novel crystal forms I and preparation method thereof |
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